Cytochemical localisation of calcium binding sites in adrenal chromaffin cells and their relation to secretion.

The ultrastructural localisation of calcium in the chromaffin cells of the adrenal medulla was investigated using the pyroantimonate technique. The electron-dense precipitate resulting from processing in the presence of pyroantimonate was removed by treatment of sections with 2 mM EGTA suggesting that the pyroantimonate technique was localising calcium since this chelator shows a 1000:1 preference for Ca2+ over Mg2+. Using dissociated cells from the bovine adrenal medulla, calcium was localised in control cells and in cells stimulated with 1 mM carbamylcholine for 1, 10 and 30 min. Stimulation resulted in an increase in plasma membrane-associated calcium within 1 min. Much of this calcium was localised at sites of granule-plasma membrane interaction and was also seen at sites of coated-vesicle formation at longer periods of stimulation. Membrane-bounded vacuoles, appearing in cells stimulated for 10 or 30 min, also showed associated electron-dense precipitate. The results obtained are consistent with a role for the calcium ion as a mediator in exocytosis at the stage of granule-plasma membrane fusion.